Isolation, characterization and kinetics of goat cystatins

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Cysteine proteinases are crucial enzymes involved in various physiological and pathological processes.
• Cystatins are endogenous inhibitors of cysteine proteinases, playing regulatory roles.
• Understanding the properties of novel cystatins can provide insights into enzyme regulation.

Purpose of the Study:

• To purify and characterize two novel cysteine proteinase inhibitors from goat kidney.
• To investigate the structural and functional properties of these inhibitors.
• To determine their inhibitory activity against relevant cysteine proteinases.

Main Methods:

• Purification using alkaline denaturation, ammonium sulfate fractionation, gel filtration (Sephadex G-75), and ion-exchange chromatography (DEAE cellulose).
• Homogeneity and molecular mass determination via SDS-PAGE under reducing and non-reducing conditions.
• Stability assessment across a range of pH and temperatures.
• Immunological identity testing with sheep low molecular weight (LMW) kininogen.
• Spectral studies (e.g., circular dichroism) to analyze secondary structure and conformational changes upon complex formation with papain.
• Enzyme inhibition assays to determine kinetic parameters (e.g., competitive inhibition, affinity) against papain, ficin, and bromelain.
• N-terminal amino acid sequencing.

Main Results:

• Two homogenous cysteine proteinase inhibitors (I and II) were purified, each with an apparent molecular mass of 67 kDa.
• The inhibitors exhibited remarkable stability, remaining active within pH 3-10 and up to 95°C.
• Immunological assays revealed identity with sheep LMW kininogen, suggesting a close relationship.
• Spectral analysis indicated a predominantly alpha-helical structure, with significant conformational changes upon papain binding.
• Both inhibitors competitively inhibited papain, ficin, and bromelain, with papain being the most potently inhibited.
• N-terminal sequencing showed maximum homology to mammalian LMW kininogen and revealed an unusual Ile substitution for Leu, potentially affecting inhibitory affinity.

Conclusions:

• Goat kidney cysteine proteinase inhibitors I and II are stable, alpha-helical proteins closely related to mammalian LMW kininogen.
• These inhibitors effectively modulate cysteine proteinase activity, particularly papain.
• The unique N-terminal sequence may influence their binding affinity compared to other known cystatins.